All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
Many childhood malignancies are lymphoid in origin and arise from transforming events that occur in early B cell progenitors [Vogler, 1978]. Understanding normal B cell development affords the opportunity to learn how the transformation process subverts normal B cell signaling mechanisms. In turn, this information provides the means to design novel targeted chemotherapeutics. In normal B cell development, rearrangement of the immunoglobulin heavy chain gene occurs during pro-B cell stages (reviewed in [LeBien, 2000]). The late pro-B cell normally is in the process of completing the rearrangement of heavy chain (V to DJ) joining. If successful gene re-arrangement occurs, then the cell will progress through the pre-B cell stage, producing IgM heavy chain (μ) protein and undergoing a burst of proliferation. Pre-B cells are identified by expression of cytoplasmic μ protein and assembly of the pre-B cell receptor (pre-BCR) complex [Loken, 1987]. If the B cell precursor fails to make a productive VDJ arrangement at both alleles, cell death will occur, which is the fate of the vast majority of (>90%) early pre-B cells [Li, 1993]. Survival, apoptotic and differentiation signals, provided by host of molecules (e.g. pre-BCR complex [Cronin, 1998], adhesion molecule receptors and cytokine receptors [Stoddart, 2000]), are tightly regulated to maintain B-lymphocyte homeostasis. An imbalance in these signals can lead to lymphoid malignancies.
The B cell at the late pro-B/early pre-B transition is a common target of transformation. In the clinical setting, acute lymphoblastic leukemia (ALL) cells derived from early B-lineage cells are loosely referred to as “pre-B ALL”, although the majority of these cells are more correctly defined as pro-B cells with no cytoplasmic μ expression.
Much work has been done to phenotypically and biochemically characterize classes of leukemia and lymphoma using a variety of models including transgenic mice [Vogler, 1978; Ichihara, 1995]. The RET protein is a tyrosine kinase expressed during the development of pro-B cells, and RET expression is down regulated when μ protein is expressed during the pre-B cell stages of B cell development [Wasserman, 1995]. Eμ-RET+ transgenic mice constitutively express activated RET tyrosine kinase under the control of the μ enhancer (Eμ), driving B-lineage restricted expression of the activated RET protein. Between 4 and 8 months of life, Eμ-RET+ transgenic mice develop lymphoblastic lymphoma/leukemia manifested by massive adenopathy, splenomegaly and bone marrow replacement [Goodfellow, 1995; Iwamoto, 1991; Wasserman, 1998; Zeng, 1998]. The malignant cells are B220+/CD431o/surface IgM−, and the majority are cytoplasmic μ- (J. Fang, unpublished data). Thus, the B-lymphoid malignancies that arise in Eμ-RET+ mice are derived from the late pro-B to early pre-B cell stage of development [Hayakawa, 1997]. The Eμ-RET+ transgenic mouse provides a developmentally targeted model of ALL that is useful in preclinical evaluation of novel therapeutic strategies.
Cytokines play an important role in promoting and controlling normal B cell development (reviewed in [Appasamy, 1999; Fry, 2002]) and are involved in malignant transformation of lymphoid precursor cells [Page, 1995]. Overall, IL-7 acts at three levels in normal lymphoid cells (reviewed in [Fry, 2001; Hofmeister, 1999; Appasamy, 1999]. It 1) acts as a trophic factor by preventing apoptosis [Lu, 1999]; 2) controls lineage-specific developmental programs such as V(D)J rearrangement [Nutt, 2001; Veiby, 1997]; and 3) stimulates proliferation of targeted cells [Corcoran, 1996]. IL-7 was originally described as a B cell growth factor secreted by bone marrow stromal cells [Henney, 1989]. Subsequently, IL-7 was found to promote the growth of pro-T cells as well, produced by cortical epithelial cells in the thymus. It is absolutely required for normal murine T and B cell development as well as human T cell development (reviewed in [Hofmeister, 1999]). IL-7 acts as a modulator of low affinity peptide-induced proliferation, a central feature of homeostatic regulation of T cell populations in humans [Fry, 2001; Tan, 2001]. Although not absolutely required for B cell development in humans, IL-7 still plays an important role in human B cell development [Pribyl, 1996; Dittel, 1995]. IL-7 provides a survival signal to early B lymphoid precursors [Smart, 2000]. IL-7 signals through the IL-7 receptor (IL-7R), a heterodimer receptor composed of two subunits, the gamma common (γc) chain (CD132) and the IL-7Rα chain (CD127) [Page, 1995]. The γc chain is shared by other cytokine receptors including IL-2R, IL-4R, IL-9R, IL-15R, and IL-21R, while the IL-7Rα chain is unique to the IL-7 receptor, whose expression varies with different stages of lymphoid development [Sudo, 1993; Armitage, 1991]. IL-7Rα chain is expressed from the early pro-B cell stage through the pre-B cell stage [Sudo, 1993]. IL-7 promotes the formation of a functional pre-B cell receptor (pre-BCR) in pro-B cells and the transition to pre-B cells. Down-regulation of IL-7 signaling in pre-B cells serves as a trigger for initiating apoptosis during negative selection of B cells with unproductive Ig rearrangements [Frishman, 1993]. PreBCR+ B cells have a proliferative advantage over PreBCR− B cells in response to low or limiting concentrations of IL-7 because of increased response to IL-7 [Fleming, 2001]. Finally, IL-7Rα expression ceases very late in the late pre-B cell stage [Smart, 2000]. When IL-7 engages the IL-7R on pro-B cells, IL-7R tyrosine phosphorylation and PI turnover occurs, resulting in clonal proliferation [Uckun, 1991]. Because the IL-7R itself has no intrinsic kinase activity, IL-7-induced phosphorylation occurs as a result of recruiting intracellular kinases, including the JAK/STAT system [Sato, 2001; van der Plas, 1996] as well as activation of PI-3 kinase [Dadi, 1993; Dadi, 1993; Dadi, 1994] and src family kinases [Seckinger, 1994].
IL-7 has been associated with certain malignancies [Karawajex, 2000; Wuchter, 2002; Touw, 1990; Barata, 2001]). Karawajew et al., reported that IL-7 rescues T cell ALL lymphoblasts from apoptosis [Karawajew, 2000]. Wuchter, et al., found that IL-7 greatly inhibited drug-induced apoptosis in T cell ALL cells [Wuchter, 2002]. IL-7 plays a role in T-cell ALL, modulating cell cycle regulators [Touw, 1990; Barata, 2001]. IL-7 leads to the down regulation of p27kip1, causing the induction of Bc1-2 leading to proliferation of leukemic T cells [Barata, 2001]. Also, IL-7 induces progression through the cell cycle; it leads to increased expression of CyclinD2/CyclinA, upregulation of CDK4 and CDK2, and phosphorylation of Rb protein [Barata, 2001]. IL-7 may be associated with Hodgkin's disease. Foss, et al., have shown elevated serum levels in a significant number of patients with Hodgkin's disease prior to treatment [Foss, 1995]. Also, IL-7 is constitutively secreted in American EBV+ Burkett's lymphoma as well as EBV+ cell lines [Benjamin, 1994]. There are several reports of IL-7 stimulating growth of human precursor B cell ALL cells [Renard, 1995]. Although there are reports of B cell lineage ALL subclones that have had decreased dependence on IL-7 and Flt-3L secreted by the BM microenvironment [Shah, 2001], the role of these cytokines in the development or progression of progenitor B cell lymphoid malignancies has not been fully elucidated [Touw, 1990; van der Plas, 1996].
Rapamycin, a mTOR inhibitor, is a macrolide antibiotic produced by Streptomyces hygroscopicus which was originally described as an antifungal agent. It is known to inhibit the growth of fungi, including Candida albicans and Microsporum gypseum. Methods for the preparation of rapamycin and characterization of its antibiotic activity were described in U.S. Pat. No. 3,929,992. Martel et al. reported that rapamycin possesses immunosuppressive properties which are effective for controlling experimental allergic encephalitis and adjuvant arthritis (1977, Canadian Journal of Physiological Pharmacology 55:48). Rapamycin has also been shown to inhibit rejection of allograft transplantation in vivo (Calne, et al., 1989, Lancet 2:227; Morris and Meiser, 1989, Medicinal Science Research 17:609). It was found that rapamycin inhibits the induction of activation and proliferation of mature T and B cells [Kay, 1991; Sakata, 1999; Morris, 1991]. Consequently, rapamycin was approved by the FDA for use as an immunosuppressive agent after solid organ transplant ([Ettenger, 2001] and reviewed in [Saunders, 2001]). There is also evidence that mTOR inhibitors, e.g. rapamycin, may inhibit the growth of and/or induce apoptosis in a wide variety of tumor types ([Eng, 1984; Douros, 1981; Houchens, 1983] and reviewed in [Huang, 2002; Huang, 2001; Elit, 2002; Hidalgo, 2000; Garber, 2001]). It has been shown that rapamycin alone [U.S. Pat. No. 4,885,171] or in combination with picibanil [U.S. Pat. No. 4,401,653] possess antitumor properties. [Calne, 1989; Schreiber, 1991; Saunders, 2001]. The development of second generation macrolides, derivatives of rapamycin, has therefore been focused on the antitumor activity of this class of drugs.
Rapamycin inhibits the activation of the mammalian Target of Rapamycin (mTOR). mTOR is a serine/threonine kinase and functions as a sensor to ensure that the cell is in an appropriate nutritional state prior to committing to cell division [Dennis, 2001; Schmelzle, 2000]. Through its interactions with other proteins, including p70S6 kinase [Dumont, 1994; Kuo, 1992], PI-3K [Castedo, 2002], and p34cdc2, mTOR regulates several processes including cell growth, initiation and elongation of mRNA translation [Castedo, 2002; Brunn, 1997; Burnett, 1998], ribosome synthesis [West, 1998], expression of metabolism-related genes, amino acid import, autophagy, and cytoskeletal reorganization (reviewed in [Raught, 2001]). By inhibiting mTOR, rapamycin mimics growth factor withdrawal, characterized by cell cycle arrest at G1 and inhibition of protein synthesis [Chen, 1994; Brown, 1994]. Upon entering the cell, rapamycin must bind to the FK-binding Protein12 (FKBP12) in order to be active [Chen, 1994; Brown, 1994]. It is this FKBP12/rapamycin complex that blocks mTOR activity [Chen, 1994; Brown, 1994]. In T cells, rapamycin shifts the balance between activation and inhibition of cyclin-dependent kinases (CDK) towards inhibition by blocking the down-regulation of p27kip1 [Nourse, 1994].
IL-2 is an important growth factor for T cells. IL-2 selectively phosphorylates p70S6 kinase [Kuo, 1992]. P70S6 kinase activates ribosomal proteins S6 and S17 to promote protein synthesis [Dumont, 1994; Kuo, 1992], and p70S6 kinase's activity is inhibited by rapamycin [Price, 1992; Frost, 1996; Patel, 1996]. IL-2 normally activates cyclin E/Cdk2 complexes by eliminating p27kip1 [Nourse, 1994]. This down-regulation of p27kip1 is key to IL-2-driven cell cycle progression. Thus, rapamycin causes inactivation of p70S6 kinase, cyclin E, Cdk2, and p34cdc2 [Kuo, 1992; Nourse, 1994; Terada, 1993; Flanagan, 1993]. Cells with low levels of p27kip1 are resistant to rapamycin, and T cells from p27kip1−/− knockout mice exhibit a significant resistance to rapamycin inhibition [Luo, 1996]. Thus, rapamycin inhibits mTOR and subsequently inhibits protein synthesis as well as cell cycle progression at the G1 to S transition. In addition to affecting IL-2 mediated signaling, rapamycin blocks the IL-7 mediated down-regulation of p27kip1 and in vivo phosphorylation of Rb protein in leukemic T cells [Barata, 2001; Ponce-Castaneda, 1995]. The persistent expression of p27kip1 in rapamycin-treated normal and leukemic T cells suggests that mTOR is a critical component of the signaling pathway that targets p27kip1 for ubiquitin-dependent proteolysis [Pagano, 1995; Morice, 1993]. Although not as well studied as in T cells, rapamycin has growth inhibitory effects in B cells in vitro [Wicker, 1990; Sakata, 1999]. Rapamycin inhibits secretion of sCD23, an autocrine B cell growth factor [Degiannis, 1995]. Crosslinking of BCR leads to p70S6 kinase activation, triggering protein synthesis via activation of ribosomal proteins [Li, 1999]. Calastretti, et al., compared rapamycin treated human follicular B cell lymphoma cell lines (characterized by high levels of BCL-2 in a steady state) to cell lines with lower BCL-2 expression level, and they found that cell lines with high expression of BCL-2 showed more inhibition than cell lines with low BCL-2 expression [Calastretti, 2001]. Harada, et al., found that p70S6 kinase phosphorylates BAD, inactivating it [Harada, 2001].